Off-target molecular recognition of cyproterone acetate by human lysozyme: conformational remodelling, enzyme...

Cyproterone acetate (CPA), a synthetic steroidal antiandrogen, is widely used in clinical therapy. However, its off-target interactions with non-receptor proteins remain inadequately explored. Here, we investigated the molecular recognition of CPA by human lysozyme (HuL), a model globular protein for folding and aggregation studies, using a combination of spectroscopic, computational, enzymatic, and aggregation assays. UV-visible absorption, fluorescence quenching and molecular docking analyses demonstrated that CPA binds HuL with moderate affinity (∼10⁴ M^-1), predominantly via hydrophobic contacts complemented by a stabilizing hydrogen bond with Asn27. This interaction induced conformational alteration, including partial unfolding and reduced β-sheet content, while preserving the overall fold. Functional assays revealed that CPA acts as a positive allosteric modulator, enhancing catalytic turnover with only a modest reduction in substrate affinity. Strikingly, biophysical and imaging studies (Thioflavin T fluorescence, light scattering, and TEM) showed that CPA significantly inhibited amyloid fibril formation, redirecting HuL into non-fibrillar, amorphous aggregates. Molecular docking further identified residue-level interactions in aggregation-prone regions, offering a structural basis for amyloid inhibition. Collectively, these findings establish CPA as an off-target modulator of protein structure, activity, and aggregation, extending its pharmacological profile beyond androgen receptor antagonism. The dual effects of enzymatic activation and amyloid suppression highlight the potential of steroidal scaffolds as anti-amyloid agents, with broader implications for protein misfolding disorders.